In vitro analysis of intra-Golgi vesicular traffic has uncovered many of the molecules involved in the process of transport vesicle budding and assembly. However, limitations of this assay have made analysis of transport vesicle binding and fusion more difficult. One aim of this proposal is to evaluate specific modifications to the conventional in vitro transport assay. These refinements will create a two-stage reaction where intra-Golgi transport vesicles bind to acceptor Golgi membranes without fusion, then fuse upon the addition of cytosolic regulatory factors. The improved assay will be used to identify proteins necessary for the specific reaction steps of binding and fusion. Two previously identified cytosolic activities, termed fractions 1-alpha and 1-beta have been found to be necessary for the combined binding and fusion assay. These activities will be further purified and characterized with respect to their specific site of action. Finally, an additional cytosolic activity, called releasing factor, will be purified and its role in binding and/or fusion determined.